Flower color is an important feature for the appreciation and purchasing of ornamental flowers, and flowers with a large variety of colors have traditionally been bred. It is rare for a single species to possess flowers of all colors, as the biosynthesis of pigments that appear as flower colors is genetically determined. Because the gene sources that can be used in hybridization breeding are limited to crossable related varieties, it is essentially impossible to produce flowers of all colors in a target variety by hybridization breeding. Recently, gene recombination techniques have made it possible to obtain flower pigment-synthesizing genes from certain plants and express those genes in different species in order to achieve modified flower color (Plant Cell Physiol. 39, 1119 (1998), Curr. Opin. Biotechnol. 12, 155 (2001)).
The flower colors of orange, red, violet and blue are exhibited primarily by flavonoids known as anthocyanins. Yellow colors generally derive from non-flavonoid compounds such as carotenoids and betalains, but the yellow colors of some plant species are due to flavonoids. For example, yellow carnations are known to possess 4,2′,4′,6′-tetrahydroxychalone (hereinafter, THC) 2-glucoside in their flower petals (Phytochemistry 5, 111 (1966)). THC 4′-glucoside is also found in Antirrhinum majus and Linaria bipartita. 
Chalcones such as, THC, butein, isoliquiritigenin and their glycosilated derivatives are known; for example, the aglycon of the glucosides in carnations, morning glory, peony, aster, strawflower, periwinkle, cyclamen and petunia is THC, in Antirrhinum majus (snapdragon) and statice it is 3,4,2′,4′,6′-pentahydroxychalcone (PHC), in cosmos and Jerusalem artichoke it is butein, and in dahlia it is butein and isoliquiritigenin. Also, certain limited species such as snapdragon, Linaria bipartita (toadflax) and morning glory contain yellow flower pigments known as aurones, including aureusidin (hereinafter, AU) and bracteatin.
Because the absorption maxima for aurones are between 399 and 403 nm, compared to absorption maxima between 372 and 382 nm for chalcones, their color tones differ and the fluorescence emitted gives aurones a sharper yellow color (Biohorti 1, 49-57 (1990), Seibundo Shinkosha). Chalcones, aurones and anthocyanins usually accumulate as glucosides in the vacuoles of plant cells. The biosynthetic pathway of anthocyanins has been thoroughly studied, and the enzymes involved in anthocyanin synthesis and their coding genes are known (Comprehensive Natural Products Chemistry, vol I (ed. Sankawa) pp 713-748, Elsevier, Amsterdam (1999)).
The biosynthetic pathway of flavonoids is widely distributed among the higher plants and is conserved among species. THC is biosynthesized from three molecules of malonyl CoA and one molecule of coumaroyl CoA, by the catalytic action of chalcone synthase. THC exhibits a light yellow color, but in plant cells it is usually rapidly converted to colorless naringenin by chalcone isomerase (CHI). Also, THC is highly unstable at near neutral pH and is converted to naringenin by spontaneous ring closure. For THC to exist stably in plant cells, i.e. for it to stably exhibit a yellow color, the 2′-position of THC must be modified with a saccharide to prevent its ring closure. The reaction is catalyzed by an enzyme that transfers glucose to the 2′-position of THC (UDP-glucose: 4,2′,4′,6′-tetrahydroxychalcone 2′-glucosyltransferase, hereinafter abbreviated as 2′CGT).
THC 2′-glucoside is present in carnation and cyclamen, and therefore 2′CGT is also predicted to be found in their flowers. Thus, it was conjectured that if the 2′CGT gene could be obtained and the enzyme gene expressed in a plant, it should be possible to accumulate THC 2′-glucosides and produce yellow flowers (Biotechnology of Ornamental Plants, Edited by Geneve, Preece and Merkle, pp 259-294, CAB International Wallingford, UK (1997)). Moreover, it was discovered that adequately accumulating THC 2′-glucoside and exhibiting yellow color requires deletion of the CHI gene to suppress enzymatic conversion from THC to naringenin, and that a clear yellow color also requires deletion of the gene for flavanone 3-hydroxylase (hereinafter abbreviated as F3H) in addition to the CHI gene (Plant Cell Physiol. 43, 578 (2002)).
While cloning of the carnation 2′CGT gene has been reported to date (Plant Cell Physiol. 44, s158 (2003)), its sequence has not been published. Also, the gene coding for 2′CGT activity has been obtained from carnation and expressed in petunia, thereby accumulating THC 2′-glucoside in petunia petals (PCT/JP03/10500). However, the THC 2′-glucoside produced by 2′CGT does not have a chemical structure that can serve as a precursor for aurone synthesis. Also, as mentioned above, accumulation of THC 2′-glucoside results in only light yellow petals.
It is known that faint yellow petals are produced by accumulation of THC having the 2′-hydroxyl methylated, but the nature of the enzyme that catalyzes this methylation and of its gene is unknown. Yellow varieties such as dahlia and cosmos contain 6′-deoxychalcone. In legumes, 6′-deoxychalcone is the precursor of 5-deoxyflavonoid, which is synthesized by the catalytic action of chalcone synthase (CHS) and chalcone reductase (CHR). It has been reported that introduction of the alfalfa CHR gene into petunia produced 6′-deoxychalcones such as butein, and that when the CHR gene was introduced into white flower petunia the flowers were mostly white upon blooming although a very light yellow color was observed at the budding stage, and therefore it was not possible to create an industrially useful yellow flower (Plant J. 13, 259 (1998)).
Because aurones exhibit a more brilliant yellow color than chalcone glucosides as explained above, it would be highly useful, for the industry, to develop a method for accumulating aurones. AS, one of the enzymes involved in aurone biosynthesis, and its gene, have already been reported in the literature (Science, 290, 1163 (2000)). According to this report, AS produces AU, bracteatin and their glucosides from THC, PHC and their glucosides as substrates. However, use of the AS gene to produce and accumulate aurones such as AU and bracteatin has not been described.
The present inventors have constructed a binary vector having the AS gene linked downstream from a structural promoter and introduced the AS gene into petunia and torenia by the Agrobacterium method, but no accumulation of aurones was observed. It has also been reported that 3-glucosylation of anthocyanidin is essential for transportation of anthocyanins into vacuoles (Nature 375, 397 (1995)), suggesting that glucosylation of aurones is likewise necessary as a transportation signal into vacuoles. In fact, the major aurone that accumulates in yellow Antirrhinum majus flower petals is AU 6′-glucoside. A GT exhibiting AU 6′-glucosylating activity (AU6GT) (WO 00/49155) was therefore obtained, and the AU6GT gene was constitutively expressed in petunia together with the AS gene, but accumulation of aurones was not observed.
The enzymes involved in flavonoid and anthocyanin biosynthesis are believed to localize in the cytoplasm or endoplasmic reticulum of cells. By the actions of these enzymes, flavonoids and anthocyanins are synthesized and glucosylated outside of vacuoles, i.e. in the cytoplasm, and transported into the vacuoles (Natural Product Reports 20, 288, (2003)). However, ardent research led the present inventors to the finding that AS is exceptional in that it localizes in the vacuoles. Thus, it was hypothesized that glucosylated chalcones may be transported to the vacuoles in vivo and used as substrates for synthesis of aurones in the vacuoles.
As mentioned above, AU 6′-glucoside is the major aurone that accumulates in the vacuoles of yellow Antirrhinum majus petals. The 6′ position of AU corresponds to the 4′ position of THC, and THC 4′-glucosides are also present in yellow Antirrhinum majus petals. On this basis, an aurone synthetic pathway was inferred wherein glucosylation of the 4′ position of THC synthesized in the cytoplasm is followed by transport to the vacuoles, and this is used as substrate for synthesis of AU 6′-glucosides by AS. Thus, it was concluded that synthesis of THC 4′-glucoside is essential for synthesis of aurones such as AU 6′-glucoside in different plant varieties. For this purpose, UDP-glucose: 4,2′,4′,6′-tetrahydroxychalcone 4′-glucosyltransferase (hereinafter, 4′CGT) is required for 4′-glucosylation of THC, and therefore the 4′CGT gene must be obtained. However, cloning of the 4′CGT gene has not yet been reported, nor do reports exist of isolating 4′CGT.
Enzymes that catalyze glucosylation of a variety of compounds including flavonoids to produce glucosides are generally referred to as glucosyltransferases (GT), and plants possess a large diversity of GT molecules and their coding genes, corresponding to the types of substrates and transferred sugars. Because GT enzymes usually utilize UDP-glucose as the glucose donor, they contain in their amino acid sequence a motif that binds UDP-glucose (Plant J. 19, 509 (1999)). Already, GT genes carrying this motif are known in 99 species of Arabidopsis whose entire genome structure has been elucidated (J, Biol. Chem, 276, 4338, (2001)).
GT enzymes and amino acid sequences and functions have also been worked out in several other plants. The genes for enzymes catalyzing reactions of transferring sugars to the 3-hydroxyl groups of flavonoids or anthocyanidins (UDP-glucose:flavonoid 3-glucosyltransferase, hereinafter: 3GT) have been obtained from perilla, corn, gentian, grape and the like (J. Biol. Chem. 274, 7405 (1999); J. Biol. Chem. 276, 4338, (2001)). In addition, genes for enzymes catalyzing reactions of transferring sugars to the 5-hydroxyl groups of anthocyanins (UDP-glucose:anthocyanin 5-glucosyltransferase, hereinafter: 5GT) have been obtained from perilla, verbena and the like (J. Biol. Chem., 274, 7405, (1999)).
Analysis of the amino acid sequences of 3GT and 5GT has shown that GT enzymes with the same function have similar amino acid sequences even in different plant varieties, or in other words, that they constitute a family (J. Biol. Chem. 276, 4338, (2001)). Thus, it is not difficult to obtain enzymes having the same function as known GT enzymes (i.e., orthologs) from other plant varieties, given the current level of technology. For example, the petunia 5GT gene has been cloned using the perilla 5GT gene (Plant Mol Biol. 48, 401 (2002)). However, much laborious trial and error is required to obtain a novel GT gene having absolutely no known ortholog.
As regards Arabidopsis whose entire genome structure is known as mentioned above, its flower petals are white and accumulation of chalcone 4′-glucosides has not been reported. Consequently, Arabidopsis GT gene information cannot be used for cloning of the 4′CGT gene. Moreover, even though 2′CGT has been isolated from carnation (PCT/JP03/10500), high homology does not necessarily exist between the 4′CGT gene and the 2′CGT gene. This is because the biochemical and molecular biological features of each GT may differ substantially if the position of sugar addition is different, even if the substrate is the same. This is also supported by the fact that 3GT and 5GT belong to different GT families. Also, betanidine 5GT and 6GT have the same substrates, and yet their amino acid homology has been reported to be only 19% (Planta 214, 492 (2002)).
In fact, the GT enzymes that transfer sugars to the 3-, 5- and 3′-positions of the same anthocyanidin skeleton belong to different families in the GT superfamily, and the amino acid homology between these families is no more than about 20% (Plant Physiol. 132, 1652, (2003), Natural Product Reports 20, 288, (2003)). Several methods are possible for obtaining not only the 4′CGT gene but also novel genes. For example, the genes for enzymes expressed in flower petals, which are involved in the synthesis of rose scent components, have been extensively sequenced and have been identified by their structures, expression patterns and expression in E. coli (Plant Cell. 14, 2325 (2002)). In order to identify the 4′CGT gene, 5,000 clones were randomly selected from a cDNA library derived from the petals of yellow Antirrhinum majus (Butterfly Yellow variety) which accumulates aurone and chalcone 4′-glucosides, and their nucleotide sequences were determined.
As a result of homology search using a public DNA database, three different GT genes were obtained. Two of the genes were the 3GT gene and the aforementioned AU6GT-coding gene (WO 00/49155), while the remaining gene was for a novel GT (designated as pSPB662) (SEQ ID NO: 13). However, the GT encoded by pSPB662 exhibited no glucosylating activity for THC, and was clearly not 4′CGT. Furthermore, as mentioned above, high expression of this gene together with the AS gene in petunia resulted in no observable production of chalcone glucosides or aurones, nor was any change in flower color seen. These results suggest that a chalcone glucosylating enzyme gene cannot be isolated by random screening of approximately 5,000 clones, and it was therefore difficult to obtain a 4′CGT gene.
Patent document 1: PCT/JP03/10500
Patent document 2: WO 00/49155
Non-patent document 1: Plant Cell Physiol. 39, 1119 (1998)
Non-patent document 2: Curr. Opin. Biotechnol. 12, 155 (2001)
Non-patent document 3: Phytochemistry 5,111 (1966)
Non-patent document 4: Biohorti 1 49-57 (1990) SEIBUNDO SHINKOSHA
Non-patent document 5: Comprehensive Natural Products Chemistry, vol I (ed. Sankawa) pp 713-748, Elsevier, Amsterdam (1999)
Non-patent document 6: Biotechnology of Ornamental Plants, Edited by Geneve, Preece and Merkle, pp 259-294, CAB International Wallingford, UK (1997)
Non-patent document 7: Plant Cell Physiol. 43, 578 (2002)
Non-patent document 8: Plant Cell Physiol. 44, s158 (2003)
Non-patent document 9: Plant J. 13, 259 (1998)
Non-patent document 10: Science, 290, 1163 (2000)
Non-patent document 11: Nature 375, 397 (1995)
Non-patent document 12: Natural Product Reports 20, 288, (2003)
Non-patent document 13: Plant J. 19, 509 (1999)
Non-patent document 14: J. Biol. Chem. 276, 4338, (2001)
Non-patent document 15: J. Biol. Chem. 274, 7405 (1999)
Non-patent document 16: Plant Mol Biol. 48, 401 (2002)
Non-patent document 17: Planta 214, 492 (2002)
Non-patent document 18: Plant Physiol. 132, 1652, 2003 (2003)
Non-patent document 19: Plant Cell. 14, 2325 (2002)